A crucial event in the formation of atherosclerotic tissues is excessive proliferation of vascular smooth muscle cells (SMC). Nitrogen monoxide (NO) provided by NO donors has been reported to be beneficial for inhibition of SMC proliferation by reducing polyamine production, activating the NO-cGMP signal pathway and neutralizing oxidative stress. Clinically approved NO donors are nitrate compounds, which are known to induce nitrate tolerance. Experimental and commercially available NO-donors exhibit relatively short half- lives. N-nitroso NO donors recently reported by us release NO in a sustained and controlled fashion with tunable rates. The apparent half-lives of these compounds ranged from 39h to 88h. We have prepared 3 new families of NO donors. The NO release profile of each of the NO-donors can be varied by changing the nature of the moieties attached to the N-nitroso group. Data obtained from cell culture studies with human aortic smooth muscle cells (HASMC), using one of the N-nitroso NO donors (80 pM) exhibited a significant (40%) decrease of SMC proliferation. More importantly, this inhibition was achieved at a very low NO-donor concentration compared to the conventional NO-donors. The proposed studies will address the following issues. First, N-nitroso NO donors (water soluble, water insoluble, low molecular weight, dendritic, pegylated, hybrid, polymeric) will be synthesized and their NO-release profiles will be determined to establish structure- activity relationships. Second, the physiological effect on HASMC of NO released by these novel NO donors will be evaluated by a variety of cell and biochemistry assays including cell viability assay, [3H] thymidine incorporation assay, and determination of arginase, nitric oxide synthase (NOS) and ornithine decarboxylase (ODC) enzyme activities. cGMP and polyamine levels in the cell culture will be determined to evaluate the effect of NO in cultured HASMC. Third, a combination arginase inhibitor, ABH, and N-nitroso NO donors will be used to achieve the "right" physiological NO level in cultured HASMC. Fourth, these combination doses will be used along with elevated level of asymmetric dimethylarginine (ADMA) to determine the beneficial effects of this combination protocol in comparison to those observed with ABH and NO-donor alone. Thus our proposed studies should provide further understanding of the effects of NO released in a slow, sustained and rate-tunable manner from the N-nitroso NO donors on HASMC as well as other SMC, cancer and neuronal cells. PUBLIC HEALTH RELEVANCE: This proposal will involve syntheses of a variety of N-nitroso NO donors and investigate their inhibition of SMC proliferation as well as the influence of structure on NO release profiles. Results of this study will benefit the use of NO donors for the prevention, diagnosis and treatment of human diseases strongly associated with impaired NO production, including cardiovascular disease, neurodegenerative diseases, chronic inflammatory diseases, and cancer.